Haynes Lab:Notebook/CRISPR Editing/2014/08/06: Difference between revisions

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* Genomic DNA extractions
* Genomic DNA extractions
 
* qPCR: CRISPR on chromatin & mCherry (single plate)




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* HEK = Gal4EED/luc cells
* HEK = Gal4EED/luc cells
* U2OS = KAH154 CRISPR cells
* U2OS = KAH154 CRISPR cells
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Sample'''
| align="center" style="background:#f0f0f0;"|'''OD 260'''
| align="center" style="background:#f0f0f0;"|'''260/280'''
| align="center" style="background:#f0f0f0;"|'''ng/uL'''
| align="center" style="background:#f0f0f0;"|'''Average ng/uL'''
|-
| Genomic DNA 7 (gRNA1d)||1.523||1.9||76.1||76.0
|-
| Genomic DNA 7 (gRNA1d)||1.514||1.88||75.7||
|-
| Genomic DNA 7 (gRNA1d)||1.526||1.89||76.3||
|-
| Genomic DNA 8 (gRNA2d)||0.315||1.79||15.7||15.6
|-
| Genomic DNA 8 (gRNA2d)||0.321||1.74||16.1||
|-
| Genomic DNA 8 (gRNA2d)||0.302||1.75||15.1||
|-
| Genomic DNA 9 (gRNA4d)||2.092||1.94||104.6||104.4
|-
| Genomic DNA 9 (gRNA4d)||2.083||1.94||104.2||
|-
| Genomic DNA 9 (gRNA4d)||2.087||1.93||104.4||
|-
| Genomic DNA 10 (-g)||1.889||1.94||94.5||94.0
|-
| Genomic DNA 10 (-g)||1.882||1.93||94.1||
|-
| Genomic DNA 10 (-g)||1.87||1.94||93.5||
|-
| Genomic DNA 1||0.772||1.93||38.6||39.0
|-
| Genomic DNA 1||0.782||1.89||39.1||
|-
| Genomic DNA 1||0.784||1.83||39.2||
|-
| Genomic DNA 2||2.17||1.96||108.5||108.2
|-
| Genomic DNA 2||2.178||1.95||108.9||
|-
| Genomic DNA 2||2.146||1.95||107.3||
|-
| Genomic DNA 3||1.016||1.89||50.8||50.6
|-
| Genomic DNA 3||1.02||1.9||51||
|-
| Genomic DNA 3||1.001||1.94||50.1||
|-
| Genomic DNA 4||1.398||1.91||69.9||68.7
|-
| Genomic DNA 4||1.35||1.93||67.5||
|-
| Genomic DNA 4||1.376||1.9||68.8||
|-
| Genomic DNA 5||4.144||1.99||207.2||206.7
|-
| Genomic DNA 5||4.179||2||209||
|-
| Genomic DNA 5||4.08||2||204||
|-
| Genomic DNA 6||4.907||1.99||245.4||244.0
|-
| Genomic DNA 6||4.9||1.98||245||
|-
| Genomic DNA 6||4.829||1.99||241.5||
|}
----
'''qPCR: CRISPR on chromatin & mCherry'''
'''qPCR Master Reaction list'''
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Reaction'''
| align="center" style="background:#f0f0f0;"|'''Templates'''
| align="center" style="background:#f0f0f0;"|'''Primers (Targets)'''
| align="center" style="background:#f0f0f0;"|'''Amplicon'''
| align="center" style="background:#f0f0f0;"|'''Method'''
|-
| 1||g20 +dox||g020US-F/ g020US-R||152||CNV
|-
| 2||g20 +dox||GAPDH B2||||CNV
|-
| 3||g20 -dox||g020US-F/ g020US-R||152||CNV
|-
| 4||g20 -dox||GAPDH B2||||CNV
|-
| 5||g29 +dox||g029US-F/ g029US-R||141||CNV
|-
| 6||g29 +dox||GAPDH B2||||CNV
|-
| 7||g29 -dox||g029US-F/ g029US-R||141||CNV
|-
| 8||g29 -dox||GAPDH B2||||CNV
|-
| 9||+dox||g020US-F/ g020US-R||152||CNV
|-
| 10||+dox||g029US-F/ g029US-R||141||CNV
|-
| 11||+dox||GAPDH B2||||CNV
|-
| 12||-dox||g020US-F/ g020US-R||152||CNV
|-
| 13||-dox||g029US-F/ g029US-R||141||CNV
|-
| 14||-dox||GAPDH B2||||CNV
|-
| 15||1d||q2F / q3R||187||CNV
|-
| 16||1d||GAPDH B2||||CNV
|-
| 17||2d||q1F / q1R||142||CNV
|-
| 18||2d||GAPDH B2||||CNV
|-
| 19||4d||q2F / P2R||140||CNV
|-
| 20||4d||GAPDH B2||||CNV
|-
| 21||mock||q2F / q3R||187||CNV
|-
| 22||mock||q1F / q1R||142||CNV
|-
| 23||mock||q2F / P2R||140||CNV
|-
| 24||mock||GAPDH B2||||CNV
|}
* 72 wells
'''Primer Master Mixes'''
# g020US-F/ g020US-R (12 wells)
# g029US-F/ g029US-R (12)
# q2F / q3R (6)
# q1F / q1R (6)
# q2F / P2R (6)
# GAPDH B2 (30)
'''Template Master Mixes'''
# g20+dox (6 wells)
# g20-dox (6)
# g29+dox (6)
# g29-dox (6)
# +dox (9)
# -dox (9)
# 1d (6)
# 2d (6)
# 4d (6)
# mock (12)
Notes:
* Opaque white plates
* Final reaction volumes = 14 μL
* Each reaction had 3.0 μL of 750 nM F/R primers, 50 ng template DNA, 1x Roche SYBR
* Batch (master) mixes...
** Primers: Primers plus SYBR, 10.5 μL mix per relevant well, one mix per unique primer pair
** Templates: DNA plus water, 4.5 μL mix per relevant well, one mix per unique template
** Final triplicates for the 96-well plate were first made as a single batch, then aliquoted from the batch tube into the 96-well plate
* previous nTc for g020US-F/ g020US-R,  g029US-F/ g029US-R, q2F / q3R, q1F / q1R, q2F / P2R, and GAPDH B2 confirmed no background signal under identical conditions
'''Run PCR - Bio-Rad CFX96'''
* 95°C, 3 min
* '''40x''' [95°C, 10 sec / 57°C, 10 sec / 72°C, 10 sec (measure)]
* 72°C, 30 sec
* Melt: 70°C -- '''+0.1°C'''/ 5 sec (measure) --> 95°C




Revision as of 08:24, 7 August 2014

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08/06/2014

  • Genomic DNA extractions
  • qPCR: CRISPR on chromatin & mCherry (single plate)


Genomic DNA extractions

Samples

  1. HEK gRNA20/Cas9, +dox cells
  2. HEK gRNA20/Cas9, -dox cells
  3. HEK gRNA29/Cas9, +dox cells
  4. HEK gRNA29/Cas9, -dox cells
  5. HEK mock, +dox cells
  6. HEK mock, -dox cells
  7. U2OS 1d
  8. U2OS 2d
  9. U2OS 4d
  10. U2OS mock
  • HEK = Gal4EED/luc cells
  • U2OS = KAH154 CRISPR cells


Sample OD 260 260/280 ng/uL Average ng/uL
Genomic DNA 7 (gRNA1d) 1.523 1.9 76.1 76.0
Genomic DNA 7 (gRNA1d) 1.514 1.88 75.7
Genomic DNA 7 (gRNA1d) 1.526 1.89 76.3
Genomic DNA 8 (gRNA2d) 0.315 1.79 15.7 15.6
Genomic DNA 8 (gRNA2d) 0.321 1.74 16.1
Genomic DNA 8 (gRNA2d) 0.302 1.75 15.1
Genomic DNA 9 (gRNA4d) 2.092 1.94 104.6 104.4
Genomic DNA 9 (gRNA4d) 2.083 1.94 104.2
Genomic DNA 9 (gRNA4d) 2.087 1.93 104.4
Genomic DNA 10 (-g) 1.889 1.94 94.5 94.0
Genomic DNA 10 (-g) 1.882 1.93 94.1
Genomic DNA 10 (-g) 1.87 1.94 93.5
Genomic DNA 1 0.772 1.93 38.6 39.0
Genomic DNA 1 0.782 1.89 39.1
Genomic DNA 1 0.784 1.83 39.2
Genomic DNA 2 2.17 1.96 108.5 108.2
Genomic DNA 2 2.178 1.95 108.9
Genomic DNA 2 2.146 1.95 107.3
Genomic DNA 3 1.016 1.89 50.8 50.6
Genomic DNA 3 1.02 1.9 51
Genomic DNA 3 1.001 1.94 50.1
Genomic DNA 4 1.398 1.91 69.9 68.7
Genomic DNA 4 1.35 1.93 67.5
Genomic DNA 4 1.376 1.9 68.8
Genomic DNA 5 4.144 1.99 207.2 206.7
Genomic DNA 5 4.179 2 209
Genomic DNA 5 4.08 2 204
Genomic DNA 6 4.907 1.99 245.4 244.0
Genomic DNA 6 4.9 1.98 245
Genomic DNA 6 4.829 1.99 241.5

qPCR: CRISPR on chromatin & mCherry


qPCR Master Reaction list

Reaction Templates Primers (Targets) Amplicon Method
1 g20 +dox g020US-F/ g020US-R 152 CNV
2 g20 +dox GAPDH B2 CNV
3 g20 -dox g020US-F/ g020US-R 152 CNV
4 g20 -dox GAPDH B2 CNV
5 g29 +dox g029US-F/ g029US-R 141 CNV
6 g29 +dox GAPDH B2 CNV
7 g29 -dox g029US-F/ g029US-R 141 CNV
8 g29 -dox GAPDH B2 CNV
9 +dox g020US-F/ g020US-R 152 CNV
10 +dox g029US-F/ g029US-R 141 CNV
11 +dox GAPDH B2 CNV
12 -dox g020US-F/ g020US-R 152 CNV
13 -dox g029US-F/ g029US-R 141 CNV
14 -dox GAPDH B2 CNV
15 1d q2F / q3R 187 CNV
16 1d GAPDH B2 CNV
17 2d q1F / q1R 142 CNV
18 2d GAPDH B2 CNV
19 4d q2F / P2R 140 CNV
20 4d GAPDH B2 CNV
21 mock q2F / q3R 187 CNV
22 mock q1F / q1R 142 CNV
23 mock q2F / P2R 140 CNV
24 mock GAPDH B2 CNV
  • 72 wells


Primer Master Mixes

  1. g020US-F/ g020US-R (12 wells)
  2. g029US-F/ g029US-R (12)
  3. q2F / q3R (6)
  4. q1F / q1R (6)
  5. q2F / P2R (6)
  6. GAPDH B2 (30)


Template Master Mixes

  1. g20+dox (6 wells)
  2. g20-dox (6)
  3. g29+dox (6)
  4. g29-dox (6)
  5. +dox (9)
  6. -dox (9)
  7. 1d (6)
  8. 2d (6)
  9. 4d (6)
  10. mock (12)


Notes:

  • Opaque white plates
  • Final reaction volumes = 14 μL
  • Each reaction had 3.0 μL of 750 nM F/R primers, 50 ng template DNA, 1x Roche SYBR
  • Batch (master) mixes...
    • Primers: Primers plus SYBR, 10.5 μL mix per relevant well, one mix per unique primer pair
    • Templates: DNA plus water, 4.5 μL mix per relevant well, one mix per unique template
    • Final triplicates for the 96-well plate were first made as a single batch, then aliquoted from the batch tube into the 96-well plate
  • previous nTc for g020US-F/ g020US-R, g029US-F/ g029US-R, q2F / q3R, q1F / q1R, q2F / P2R, and GAPDH B2 confirmed no background signal under identical conditions


Run PCR - Bio-Rad CFX96

  • 95°C, 3 min
  • 40x [95°C, 10 sec / 57°C, 10 sec / 72°C, 10 sec (measure)]
  • 72°C, 30 sec
  • Melt: 70°C -- +0.1°C/ 5 sec (measure) --> 95°C




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